The objective of this research program is to evaluate the putative role of mitogen receptors in regulating in vitro and in vivo replication by characterizing insulin-like activity (ILAS) and insulin binding to substratum-attached chick embryo fibroblasts (CEF), RSV-transformed CEF, and to isolated plasma membrane from these cells. ILAs with a pI of 6.4-6.7 and MW of 9-10,000 daltons was prepared from acid-ethanol extracts of human plasma by Sephadex and CMC chromatography. This polypeptide which is similar in its biophysical properties to somatomedin A stimulates TdR incorporation into the DNA of confluent cultures of CEF and 3T3 cells at a rate that was one-half that achieved with 10 percent fetal calf serum (FCS). 125I-ILAs binding to substratum-associated CEF was characterized in general by incubating CEF on 60 mm culture dishes with 1 ml PBS-3 percent BSA containing 5 micron U (0.2 ng) of radiolabelled ligand. Specific binding is the difference between total binding and binding in the presence of 250 micron U of unlabelled ILAs or 100 micron g of insulin. At 42 degrees C, ILAs binding was proportional to protein concentration, maximum by 60-90 min and optimum at pH 8.2. The bound ILAs dissociated completely from CEF during 120 min of incubation at 24 degrees C and the rate was not accelerated by unlabelled mitogen. ILAs binds specifically to CEF. Fifty micron U/ml of unlabelled ILAs inhibited the binding of 5 micron U of 125I-ILAs by 50 percent with an apparent Kd of 0.4 times 10 to the minus 9th power M. Insulin's inhibition of 125I-ILAs binding was 2-6,000 fold less potent. 125I-Insulin binding to CEF was inhibited by ILAs but 10-fold more was required than was needed to inhibit 125I-ILAs binding. It was, however, about 1/1000 the quantity of insulin required to achieve a similar level of inhibition of 125I-insulin binding. The binding of 125I-ILAs and 125I-insulin and the rate of incorporation of TdR were studied as functions of CEF plating density.